Project Summary/Abstract Glutamate transporters play essential roles in controlling the levels of the neurotransmitter glutamate in the mammalian brain. However, when energy supply is impaired, for example in a stroke, glutamate transporters run in reverse, releasing the excitotoxin glutamate into the extracellular space, resulting in neuronal cell death. The long-term goals of this proposal are to explore the fundamental physical principles by which these transporters release glutamate through reverse transport, and to develop methods to prevent this glutamate release. Despite progress towards these goals through functional studies, as well as the identification of structural models of a bacterial homologue of mammalian glutamate transporters in several states, basic questions about the mechanism of reverse transport, its activation by external potassium, and the pharmacology of reverse transport blockers remain unresolved. We propose two specific aims to address these important questions: Aim 1 will develop means to selectively block glutamate release by reverse transport, without blocking glutamate uptake, and will identify new paradigms of inhibition mechanism. The hypothesis will be tested that introduction of a pro-drug blocker into the cytosol will enable the specific inhibition of reverse transport by binding of the blocker to the intracellular substrate binding site. Aim 2 will determine mechanisms by which extracellular potassium activates and regulates glutamate release through reverse transport, and how potassium cooperates with sodium to form previously unknown states of the transporter. Aim 2 will also identify structural elements contributing to interaction with, and reverse translocation of K+. The proposed experiments will test the hypotheses that external K+ has an inhibitory effect on glutamate release at high concentrations, and that it can bind in the presence of Na+ to form a novel K+/Na+ co-binding state. To approach these two aims, a combination of experimental and computational methods will be used, allowing us to rapidly assess glutamate release through reverse transport, and model it using kinetic and all-atom molecular dynamics simulations. Conceptually and methodologically the proposed research is innovative, because we expect to identify novel aspects of reverse transport mechanism, as well as develop novel methods to measure and modulate the rate of reverse transport, based on extensive preliminary data. Understanding the physical principles underlying glutamate release through reverse transport, as well as its modulation by inhibitors, is important because it will contribute to our knowledge of the role of glutamate transport in the normally-functioning brain. In addition, the expected results could be ultimately used to extend existing, or devise new strategies, to reduce the destructive role of glutamate release through glutamate reverse transport in neurodegenerative disease and stroke.